The adaptive immune system is a highly evolved, flexible system for the recognition and neutralization of foreign organisms and macromolecules. At the core of adaptive immunity is an engine for the creation of a vast variety of different similar structures that have been diversified by combinatorial assembly of varied building blocks with highly random linker segments. The two principle recognition complexes of the higher vertebrate adaptive immune system, antibodies and the T cell antigen receptor, are similarly assembled, and function through their cognate cell types, B cells and T cells, to produce a coordinated resistance to pathogens. Although all elements of the adaptive recognition system of higher vertebrates are based on assemblies of monomer domains of the immunoglobulin fold, in cyclostomes, convergent evolution has created an adaptive immune system that is constructed by the assembly of recognition elements derived from leucine rich repeats.
The effector proteins of the B cell arm of the adaptive immune system, particularly antibodies of the IgG subtype, have many attractive properties as candidate therapeutic agents. IgG antibodies are highly soluble proteins with a long in vivo half-life that have weak immunogenicity within a given species. They often can be selected to have high affinities for their targets and are known to have few intrinsic safety liabilities. As a class IgG antibodies have relatively predictable behavior in vitro and in vivo, and in recent years recombinant antibodies of substantially human sequence have played a major role in therapeutic medicine as universal recognition moieties for a number of targets in different diseases. Human monospecific antibodies of the IgG subtype provide high specificity, bivalency, fully human composition, and long plasma half-life. The known limitations of antibodies relate largely to their biophysical properties (high molecular weight, multi-domain assemblage, disulfide bonds, glycosylation), which require eukaryotic manufacturing processes that are more complex and more expensive than their prokaryotic counterparts.
Scaffolds based on different human or non-human proteins or protein domains have emerged as an independent class of alternative therapeutic molecules. The status of alternative scaffolds and selection procedures used to identify high affinity binding proteins based on those scaffolds has been reviewed (Gebauer, M. & Skerra, A. Curr. Opin. Chem. Biol. 2009, 13:245-255). Different proteins have been investigated as frameworks for bringing the diversified sequences to targets, including affibodies, lipocalins, ankyrin-repeat proteins, natural peptide binding domains, enzymes, GFP, small disulfide-bonded peptides, protease inhibitors, and others.
Although for prospective therapeutic applications to date, alternative scaffolds have largely been employed as neutralizing agents for ligand-receptor interaction, cytokine, toxin, or Fc-fusions are being investigated to confer on the binding protein a cytostatic or cytotoxic effect similar to that achieved through antibody-dependent cellular cytotoxicity (ADCC). The potential role of alternative scaffolds in diagnosis is important since large arrays of specific small reagents could be produced to many different targets. Compared to antibodies, small scaffolds should have better tissue penetration which could be advantageous for solid tumor targets (Zahnd C., et al. Cancer Res. 2010, 70(4):1595-1605).
Even though it has not heretofore been emphasized in the development of antibody-like binders using engineered scaffold proteins, the evolvability of a parent protein has been recognized as a key factor for successful directed evolution of enzymatic activities (Bloom, J. D. & Arnold, F. H, Proc. Natl. Acad. Sci., 2009, 106:9995-10000). Two evolutionary concepts have been used to provide rational basis for increased evolvability of enzymes: (i) the conservation of catalytic mechanisms, and (ii) the functional promiscuity. First, the knowledge of the catalytic motifs responsible for conserved aspects of catalysis in mechanistically diverse superfamilies could be used to identify promising templates for protein engineering. Second, protein evolutions often proceed through promiscuous intermediates, suggesting that naturally promiscuous templates (for a target reaction) could enhance protein engineering strategies (Khersonsky, O., et al., Curr. Opin. Chem. Biol. 2006, 10:498-508).
Catalysis of different chemical reactions by evolutionarily related proteins has been observed in several protein fold classes. The thioredoxin fold is found in thioredoxin superfamily of proteins that serve a wide variety of functions, including protein disulfide isomerases, DsbAs, the glutaredoxins, glutathione S-transferases, calsequestrins, and glutathione peroxidases and peroxiredoxins (Copley, S. D. et al. Biochemistry, 2004, 43:13981-13995). These proteins have been shown to interact with many different types of protein substrates, demonstrating the ability of the thioredoxin fold to recognize diverse targets. Moreover, based on combined sequence, structural, and functional evidence for homology, more than 723 proteins have been identified to possess a thioredoxin-like fold (containing different circular permutations including that of thioredoxin fold) and may be divided into at least eleven different evolutionary families (Qi, Y. & Grishin, N. V., Proteins 2005, 58:376-388). Further computational analysis revealed that the thioredoxin-like fold class, as described in Qi, Y. & Grishin, N. V., Proteins 2005, 58:376-388, is the largest sets of proteins likely to have evolved from a common ancestor, incorporating at least eighteen individual superfamilies and comprising 29,206 sequences (Atkinson, H. J. & Babbitt, P. C., PloS Comput. Biol. 2009, 5(10):e1000541). The evolutionary relationships among some of these protein families have been documented. The observed flexibility and adaptation of thioredoxin-like fold makes it extremely suitable for the construction of proteins with novel functions. Sequence alignment of proteins from the thioredoxin-like fold families also reveals the high degree of sequence variations in the fold, implying its potential to allow large numbers of mutations to be explored during directed evolution experiments for the selection of prospective binders.
Many members of the thioredoxin superfamily share two features in common: they contain a short sequence motif that includes a -CPGC- sequence (the active site) and an overall structure containing this motif that bears the same topology as thioredoxin. Laboratory evolution of the proteins with thioredoxin fold further demonstrates the flexibility of the fold and helps illustrate how various functions can be acquired by individual members that did not possess these functions prior to the imposed selection (Pan, J. L. & Bardwell, J. C., Protein Sci., 2006, 15:2217-2227). For example, substitution of thioredoxin's active site CGPC to DsbA's active site CPHC can result in a protein that functions very similarly to DsbA. Another example of in vitro evolution is the selection of thioredoxin mutants that can compensate for the whole DsbA-DsbB pathway. A mutation from CGPC to CACC in exported versions of thioredoxin was capable of complementing null mutations in the DsbA-DsbB pathway. They do so by acquiring a 2Fe-25 iron-sulfur cluster, and presumably a whole new mechanism of action. This shows that thioredoxin is extremely amenable to mutation, conferring the protein with new catalytic properties and the ability to participate in new redox reactions. Conversion of a peroxiredoxin into a disulfide reductase was accomplished by a single TCT insertion in the gene ahpC, which allowed the AhpC protein product to function as a disulfide reductase as opposed to the peroxiredoxin role that it normally participates in within the cell. AhpC has lost its peroxidase activity while gaining a disulfide reductase activity. Additionally, some multi-domain thioredoxin super family proteins contain a non-catalytic thioredoxin-like domain that involves in substrate binding (Pedone, E. et al. Cell. Mol. Life Sci., 2010, July 13, Epub ahead of print). For example, human protein disulfide isomerase (PDI) contains two catalytic domains, a and a′, and two non-catalytic domains, b and b′. Biochemical studies have established that the b′ domain is sufficient for binding small peptide substrates, even though catalytic domains a and a′ are also involved in binding of larger protein substrates. Within the b′ domain, the implicated ligand binding site is a small hydrophobic pocket located in a position homologous to that of the active site in the catalytic domains.
These examples show that thioredoxin and thioredoxin-like proteins can evolve, both in function and substrate specificity, with only a few amino acid changes in the protein. Although the function and specificity has changed, the thioredoxin fold is still conserved.
Thioredoxin (Trx) is the founding member of the thioredoxin superfamily (Martin, J. L. Curr. Biol., 1995, 3:245-250). It is a 12-kDa protein that is involved in many reactions including reducing improper disulfides that have formed in the cytosol, donating reductive equivalents to ribonucleotide reductase, and being an indicator of the intracellular redox status. The function of thioredoxin has been implicated in numerous pathways; principally, it provides a protective role against many different types of damaging stresses (Lillig, C. H. & Holmgren, A., Antioxid. Redox Signal., 2007, 9(1):25-47). In addition to its anti-oxidative effect by dithiol-disulfide exchange in its active site, Trx has anti-apoptotic and anti-inflammatory effects. Trx overexpression has been shown to be effective in a wide variety of animal models for oxidative and inflammatory disorders. An administration of recombinant Trx protein is also effective in animal models for severe acute lung diseases where Trx is likely to act with its anti-inflammatory properties (Nakamura H. et al., Adv. Drug Deliv. Rev. 2009, 61(4):303-309). Although it has no signal peptide, Trx is released from cells in response to oxidative stress. Trx is found in circulation and shows anti-chemotactic effects for neutrophils and inhibitory effects against macrophage migration inhibitory factor (MIF). Neovascularization is also suppressed by Trx via inhibition of the complement activation. The anti-inflammatory effects of Trx suggest that it is not likely to elicit immunogenic responses in vivo.
Pharmacokinetics of Trx has also been studied (Nakamura H. et al., Adv. Drug Deliv. Rev. 2009, 61(4):303-309). When recombinant human Trx was injected intravenously, its half-life in plasma was measured to be roughly 1 h in mouse, 2 h in rat, and 8 h in monkey. In healthy volunteers, Trx is circulating in plasma at the concentrations of 10-30 ng/ml and, in the kidney it is excreted through the glomerulus and mostly reabsorbed by the proximal tubules, such that Trx levels in the urine of healthy volunteers are quite low and usually undetectable. When an excess amount of Trx such as 10 mg/kg is injected into animals, Trx protein is excreted into the urine as an immunologically intact form, suggesting that this protein is not likely to be metabolized. Tissue deposition of Trx after intravenous injection was limited. Five plasma proteins were identified to interact with recombinant Trx: apolipoprotein A-I, scavenger receptor (cysteine rich domain), fibrinogen (gamma polypeptide), complement factor H, and albumin. Interaction with albumin may be particularly beneficial for prolonged half-life in plasma.